The invention relates to the development of embryos and normal offspring from fertilized enucleated recipient oocytes that have been reconstituted with polar body chromosomes. The invention further relates to the development of multiple embryos and multiple live offspring having the genetic material from a single oocyte.
The meiotic division of mammalian oocytes differs in several significant ways from that of male germ cells. In the male, as a result of meiotic divisions of single diploid cells (spermatogonia), sets of four haploid round spermatids with an equal volume of cytoplasm are formed. Each of these spermatids then develops into a spermatozoon. However, in the female, one primary germ cell (oocyte) gives rise to only one mature ovum (egg). During meiotic division of the oocyte, two small polar bodies (the first and second polar bodies), each containing a set of chromosomes and very little cytoplasm, are sequentially extruded from the large main egg body. Neither of these polar bodies serves any known function. Rather, they each degenerate sometime during the pre-implantation period and do not participate in embryonic development. Each of the polar bodies has been used for pre-implantation diagnosis of gene defects and chromosomal disorders in humans, as an alternative to embryonic biopsy diagnosis.
In normal mammalian development, oocytes become developmentally arrested in the ovaries at the germinal vesicle stage in prophase of the first meiotic division. Upon appropriate stimulation (e.g., a surge in plasma luteinizing hormone), meiosis resumes, the germinal vesicle breaks down, and the first meiotic division is completed with the extrusion of a diploid set of chromosomes into the first polar body, another diploid set of chromosomes remaining within the cytoplasm of the oocyte. The oocyte then becomes arrested at metaphase of the second meiosis ("Met II"). Met II oocytes (mature oocytes) can then be ovulated and fertilized. Once fertilized, the oocyte completes the second meiotic division with the extrusion of a haploid set of chromosomes into the second polar body, male and female pronuclei are formed, and DNA replication is initiated in the pronuclei. The male and female pronuclei then fuse together, allowing their chromosomes to mingle. Equal segregation of the genetic material occurs by mitosis and the zygote cleaves to form two daughter blastomeres. The embryos continue to develop by undergoing a series of mitotic divisions before differentiating into specific cells, resulting in the organization of tissues and organs. This developmental program ensures the successful transition from oocyte to offspring.
Recently, it has been demonstrated in the mouse that the female pronucleus can be removed from a fertilized oocyte and replaced with the second polar body chromosomes from the same or a different oocyte and, providing they are synchronized in age with the male pronuclei, the second polar body chromosomes are competent to support embryonic development resulting in live mouse offspring. The first polar body has been shown to be extremely unstable. For example, in mouse oocytes, more than half of the first polar bodies degenerate within a few hours after ovulation, and the vast majority disintegrate during the next 12 hours. In contrast, in humans it has been demonstrated that many first polar bodies persist for more than 20 hours after ovulation. In general, it is believed that the first polar body in eutherian (placental) mammals has a shorter life than the second polar body. Although degeneration of polar bodies (and of unfertilized oocytes as well) is likely to be an apoptotic process, the factors that determine the individual and species differences in the degeneration rates of polar bodies (and of unfertilized oocytes) are not understood.
It has never been determined whether the chromosomes within the first polar body have the same genetic and reproductive potential as those left within the secondary oocyte after the first meiotic division. That is, it has never been determined whether the chromosomes of the first polar body can participate in normal embryonic development and support the production of live offspring. An advantage to be gained by utilizing the first polar body chromosomes to obtain a live offspring, in addition to a live offspring produced from the normal fertilized ovum, is that two offspring could be obtained that have chromosomes (i.e. the genetic information) obtained from a single oocyte. Moreover, if second polar body chromosomes from both the normal fertilized ovum and the fertilized first polar body chromosome recipient oocyte were similarly utilized to obtain live offspring, it is theoretically possible to produce four individual offspring using the chromosomes from a single donor oocyte, providing that oocytes suitable as recipients of the chromosomes are available.
The advantages of such a method could be enormous. For example, multiple offspring could be produced that contain the genetic information from a single oocyte of a female whose genetic information it would be desirable to propagate (e.g., a zoo animal, an endangered mammalian species, a non-human primate, a human, or an animal having superior breeding, such as a race horse, livestock, and the like). Moreover, since each recipient oocyte would be fertilized by a different spermatozoon and, because the genotypes of the polar bodies may not be identical to each other or to the donor oocyte, these offspring would not be clones, thus providing the opportunity to increase the genetic diversity of the offspring.